Amphibian Metamorphosis Assay OCSPP Metamorphosis Assay . OCSPP Guideline 890.1100 . ... Amphibian...

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  • Amphibian Metamorphosis Assay OCSPP Guideline 890.1100

    Standard Evaluation Procedure (SEP)

    ENDOCRINE DISRUPTOR SCREENING PROGRAM U.S. Environmental Protection Agency

    Washington, DC 20460

    30 September 2011

  • Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100

    Table of Contents

    I. INTRODUCTION..................................................................................................... 1 II. THE AMPHIBIAN METAMORPHOSIS ASSAY ................................................ 2

    A. Purpose of the Assay ............................................................................................ 2 B. Study Design ........................................................................................................ 2

    III. EVALUATION OF STUDY CONDUCT ............................................................... 2 A. Test Species .......................................................................................................... 3 B. Equipment and Supplies ....................................................................................... 3 C. Chemical Testability ............................................................................................ 3 D. Exposure System .................................................................................................. 3

    1. System Description ........................................................................................... 3 2. Water Quality ................................................................................................... 3 3. Iodide Concentration in Test Water .................................................................. 4

    E. Holding of Animals .............................................................................................. 4 1. Adult Care and Breeding .................................................................................. 4 2. Embryo Selection ............................................................................................. 4 3. Larval Culture and Feeding .............................................................................. 4

    F. Analytical Chemistry............................................................................................ 5 G. Selection of Test Concentrations.......................................................................... 5

    1. Establishing the High Test Concentration ........................................................ 5 2. Test Concentration Range ................................................................................ 5

    H. Test Procedure ...................................................................................................... 6 1. Day 0 (Test Initiation) ...................................................................................... 6 2. Day 7................................................................................................................. 7 3. Day 21 (Test Termination) ............................................................................... 7

    I. Determination of Biological Endpoints................................................................ 8 1. Mortality and Clinical Signs ............................................................................. 9 2. Developmental Stage ........................................................................................ 9 3. Hind Limb Length (HLL) ................................................................................. 9 4. Snout-Vent Length (SVL) and Body Weight ................................................. 10 5. Thyroid Gland Histopathology ....................................................................... 10 6. Specimens Archival ........................................................................................ 11 7. Data Reporting and Completeness ................................................................. 11

    IV. STUDY INTERPRETATION ............................................................................... 11 A. Test Validity Criteria .......................................................................................... 11 B. Performance Criteria .......................................................................................... 12

  • Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100

    C. General Analysis ................................................................................................ 13 1. Statistical Analyses ......................................................................................... 13 2. Trends ............................................................................................................. 14 3. Histological Findings ...................................................................................... 15

    D. Endpoint Interpretation ...................................................................................... 15 1. Advanced Development (Developmental Stage and Normalized HLL) ........ 15 2. Asynchronous Development (Unable to Stage) ............................................. 16 3. Histopathology................................................................................................ 17 4. Developmental Delay (Developmental Stage and Normalized HLL) ............ 18 5. Growth (SVL and Body Weight).................................................................... 18

    E. Special Data Analysis Considerations................................................................ 18 1. Use of Compromised Treatment Levels ......................................................... 18 2. Solvent Controls ............................................................................................. 19 3. Treatment Groups Achieving NF Developmental Stage 60 and Above ........ 19 4. Histological Analyses and Developmental Stage ........................................... 19

    V. DATA EVALUATION RECORD ......................................................................... 19 VI. REFERENCES ........................................................................................................ 20

    Appendices

    Appendix 1: Staging ......................................................................................................... 22 Appendix 2: Length Measurement.................................................................................... 23 Appendix 3: Decision Logic for the Conduct and Interpretation of the AMA ................. 24 Appendix 4: Recommendations for Statistical Analyses Based on OECD 2006 ............. 25 Appendix 5: Expanded Bibliography................................................................................ 28

  • Standard Evaluation Procedure (SEP) Amphibian Metamorphosis Assay, OCSPP 890.1100 Page 1 of 51

    I. Introduction

    This document was developed by EPA to provide guidance to EPA staff who will be reviewing the data submitted in response to Tier 1 Orders issued under the Endocrine Disruptor Screening Program (EDSP). This document provides general guidance and is not binding on either EPA or any outside parties. The use of language such as "will," "is," "may," "can" or "should" in this document does not connote any requirement for either EPA or any outside parties. As such, EPA may depart from the guidance where circumstances warrant and without prior notice. The SEPs are intended to be used in conjunction with the EDSP Test Guideline Series 890 and the Corrections and Clarifications document available on the EDSP web page.

    This Standard Evaluation Procedure (SEP) provides guidance on how EPA generally intends to review studies conducted using the OCSPP Guideline 890.1100 Amphibian Metamorphosis (Frog) Assay (AMA) that are submitted to support requirements imposed under the U.S. Environmental Protection Agencys Endocrine Disruptor Screening Program (EDSP). The objective of EDSP Tier 1 assays is to characterize the potential of a chemical to interact with the endocrine system.

    The product of the review will be a Data Evaluation Record (DER) that reflects

    how well the study conforms to the Guideline, evaluates how well the study and analyses were performed, and provides the conclusions supported by the data. The DER will include, for example, a list of any significant deviations from the guideline and their potential impacts, a list of significant information missing from the study report, a description of how the statistical analyses were performed and whether they were performed according to the guideline, and any other information about the performance of the study that affects interpretation of the data within the context of the EDSP. The DER should record details on all endpoints required by the guideline. The DER is intended to contain enough information to provide EPA with the ability to determine whether the study is scientifically valid and provides the necessary information.

    The guideline recommends the critical materials, methods, and analyses that lead

    to successful performance of the assay. If a particular material, method, or analysis is specified in the guideline, it is usually because other materials, methods, or analyses are either known to be inappropriate, or at least have not been validated and there is concern for their potential influence on results. The Agency has posted Corrections and Clarifications on Technical Aspects of the EDSP Tier 1 Assays (OCSPP Test Guideline Series 890) in the docket; the link to this document may be found by way of the EDSP web page (http://www.epa.gov/endo/). It is therefore important to note deviations from specific materials, methods, or analyses in the DER, and provide the Agencys opinion on whether the deviation/deficiency